The present invention relates to plant gene regulatory regions and their use in the expression of genes of interest. More specifically, the present invention relates to the use of an aleurone regulatory region for organ, and tissue specific expression of a gene of interest within aleurone tissues of plants, and for constitutive expression of a gene of interest within monocot and dicotyledonous plants.
The endosperm of a seed is the site of deposition of storage products such as starch and proteins used by the developing embryo during germination. The endosperm surrounds the embryo of developing and mature cereal seeds. The endosperm comprises a peripheral layer of aleurone cells, which are specialized secretory cells. During germination, the aleurone layer is involved in the transfer of metabolites from the transport system to the endosperm. Furthermore, several antimicrobial compounds required to protect the seed during dormancy, imbibition and germination are synthesized within this tissue. The aleurone cells differentiate from primary endosperm cells 10-21 days after fertilization.
Several aspects of hormonal regulation of gene transcription within aleurone tissue, in germinating barley seeds have been well characterized (Fincher 1989, Annu. Rev. Plant Physiol. Mol. Biol. 40:305-346). For example, genes encoding .alpha.-amylase, responsible for the digestion of the starch stored within the starchy endosperm, and .beta.-glucanase, which digests the cell walls, have been isolated and characterized (WO 90/01551 Rogers; U.S. Pat. No. 5,677,474 Rogers, issued Oct. 14, 1997; Karrer et al 1991, Plant Mol. Biol. 16:797-805; Slakeski and Fincher 1992). Furthermore, several structural and regulatory genes involved in anthocyanin biosynthesis within the aleurone have been isolated and characterized (Paz-Ares et al 1987, EMBO J. 5:829-833; Dellaporta et al 1988, pp263-282 18th Stadler Genet. Symp. ed. J. P. Gustafsant and R. Appels). Other genes representing differentially expressed transcripts within aleurone layers have also been reported including CHI26 (Lea et al 1991, J. Biol. Chem. 266:1564-73); pZE40 (Smith et al 1992, Plant Mol. Biol. 20:255-66); pHvGS-1, and pcHth3 (Heck and Ho 1996, Plant Mol. Biol. 30:611-23). Several genes encoding lipid transfer proteins (Ltp) have also been obtained from barley aleurone tissues, including B11E-barley Ltp1, and B12A-barley Ltp2 (Jakobsen et al 1989, Plant Mol. Biol. 12: 285-93). Only one of these genes, B12A, has been expressed ectopically in transgenic plants. In this case the promoter is active only during seed development (Kalla et al 1994 Plant J. 6:849-860)
Lipid transfer proteins are responsible for the transfer of phospholipids between membranes in vitro, and likely play a role during membrane biogenesis. This may be especially important in aleurone cells which are known to develop extensive membrane systems. Skriver et al (1992, Plant Mol. Biol. 18: 585-589) disclose the sequence of a genomic Ltp (Ltp1), including the promoter region, from barley. Northern analysis demonstrated that this gene was specifically expressed in developing and germinaring seeds, as well as in whole seeds and aleurone layers obtained from seeds 30 days post anthesis (dpa). No expression of Ltp1 mRNA was observed in root, leaf, or shoot tissues, or coleoptiles of germinating seeds. Linnestad et al (1991, Plant Physiol 97: 841-843) also discloses the promoter sequence of the Ltp1 promoter from barley which was obtained using barley cDNA B12A as a probe. The Ltp1 promoter, as well as a modified form of this promoter is disclosed in WO 95/23230 (Feb. 23, 1995; Olsen et al). The modified form of the Ltp1 promoter was not specific to directing expression within aleurone cells, and was active in a range of plant organs and tissues including aleurone cells, scutellar epithelial tissue and vascular tissue during germination or in the plant, including root, leaves and stem.
The promoter of B12A (also termed Ltp2) directs expression specifically within the aleurone layer of developing grain as determined using transgenic cereal plants (Kalla et al 1994, Plant J. 6: 849-860). The sequence of the Ltp2 promoter is disclosed in CA 2,110,772 (filed Dec. 6, 1993, Olsen and Kalla) and U.S. Pat. No. 5,525,716 (Kalla et al). Dieryck et al (1992, Plt,. Molec. Biol 19:707-709) disclose the incomplete CDNA sequence of a wheat (Triticum durum) Ltp (pTd4.90). Ltp genes comprise a multigenic family and are ubiquitous in plants. Unfortunately as these genes or corresponding proteins have been isolated from various species there is no uniformity in the terminology used to identify the genes. Hence Ltp1 from tobacco, barley and Arabidopsis are not the same. As well, two barley Ltp2 genes are described in the literature; barley Lpt2 as described in Molina and Garcia-Olmedo (Plant J. 4:983-991) is a leaf Lpt, while barley Ltp2 as described in Kalla et al (1994 Plant J. 6:849-860) is aleurone specific.
It is desirable to provide regulatory regions capable of controlling aleurone specific expression that is not detrimental to the developing embryo and seedling. Aleurone-specific regulatory regions may be used for the regulation of the expression of heterologous or native genes within aleurone tissue of cereal seeds in order to modify grain development and germination. For example, placing genes of interest under the control of aleurone-specific regulatory regions may be used to:
1) mediate the unloading of metabolites from the transport system into the endosperm, since this metabolite unloading is processed through aleurone cells. By expressing genes of interest involved in tis process specifically within the aleurone, the grain yield may be affected. For example, which is not to be considered limiting in any manner, these genes of interest may include Na.sup.+ and K.sup.+ ATPases functioning in active transport, modifiers of membrane pore exclusion parameters such as TMV movement proteins, invertase for sucrose transport etc.; PA1 2) affect the quality of the grain, through the production of specific proteins or enzymes, lipids, secondary metabolizes etc. and their secretion into the endosperm during endosperm development or endosperm digestion. For example, which is not to be considered limiting in any manner, such proteins may include starch syntheses, ADP glucose pyrophosphorylase, monoclonal antibodies, glutenins, anticoagulants (eg hirudin), anti-pathogenic phenolics etc. Furthermore, expression of a gene of interest within the aleurone may also be used in order to express proteins for nutritional or medicinal purposes for feeding to animals or humans; PA1 3) regulate pre-harvest sprouting by affecting dormancy, for example which is not to be considered limiting, by over-expression of ACC syntheses to induce inhibitory levels of ethylene; PA1 4) enhance alcohol production--introduction of novel high temperature resistant enzymes for industrial application, including, but not limited to, thermostable amylases, pectinases and invertase; PA1 5) modify disease resistance of developing and germinating grains by expressing proteins, for example but not limited to, oxalate oxidase, glucose oxidase, chitinase, or lipid transfer proteins, in combination with a suitable signal peptide for targeting to the extracellular matrix and cell wall localization. This approach can be used to modify the matrix to provide a stronger physical barrier against invading pathogens or to direct specific anti-pathogen agents to the aleurone/pericarp interface. PA1 i) a first regulatory element comprising the nucleotide sequence of SEQ ID NO:3, or a nuclectide sequence that hybridizes to the nucleotide sequence of SEQ ID NO:3 under the following conditions: hybridization in 5.times.SSC and 50% formamide at 42.degree. C.; and washing in from about 0.5.times.SSC to about 0.2.times.SSC at 65.degree. C., and exhibits regulatory element activity; and PA1 ii) a second regulatory element comprising at least one exogenous mediator of the regulatory activity of the first regulatory element. PA1 i) the nucleotide sequence of SEQ ID NO:3 or a nucpeotide sequence that hybridizes to the nucleotide sequence of SEQ ID NO:3 under the following conditions: hybridization in 5.times.SSC and 50% formamide at 42.degree. C.; and washing in from about 0.5.times.SSC to about 0.2.times.SSC at 65.degree. C., wherein the nucleotide sequence exhibits regulatory element activity; and PA1 ii) a gene of interest in operative association with the nucleotide sequence. PA1 i) a gene of interest for which expression is desired in operative association with the chimeric regulatory region comprising a first regulatory element comprising the nucleotide sequence of SEQ ID NO:3, or a nucleotide sequence that hybridizes to the nucleotide sequence of SEQ ID NO:3 under the following conditions: hybridization in 5.times.SSC and 50% formamide at 42.degree. C.; and washing in from about 0.5.times.SSC to about 0.2.times.SSC at 65.degree. C., and exhibits regulatory element activity; and a second regulatory element comprising at least one exogenous mediator of the regulatory activity of the first regulatory element to produce a gene construct; and PA1 ii) introducing the gene construct into the plant and allowing for expression of the gene of interest. PA1 i) a gene of interest for which expression is desired in operative association with the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence that hybridizes to the nucleotide sequence of SEQ ID NO:3 under the following conditions: hybridization in 5.times.SSC and 50% formamide at 42.degree. C.; and washing in from about 0.5.times.SSC to about 0.2.times.SSC at 65.degree. C., wherein the nucleotide sequence exhibits regulatory element activity, to produce a gene construct; and PA1 ii) introducing the gene construct into the dicotyledonous plant and allowing for expression of the gene of interest.
This invention characterizes a novel wheat aleurone specific regulatory region active during embryo development and germination and which controls expression of heterologous genes of interest within transgenic plants. Furthermore, this invention relates to a constitutive regulatory element that is active within monocot and dicotyledonous plants, and which can be used to drive the expression of a gene of interest in a variety of plants.